An immunochemical reaction is here defined as the specific binding which takes place between an antigen and antibody. Antigens and antibodies are components of the immunity system whereby mammals including man protect themselves against infectious agents. An antigen is any substance foreign to the organism into which it has been introduced, which is capable of eliciting the protective immune response of that organism. Most antigens are proteinaceous materials in whole or in part, having high molecular weights. Antibodies are also proteinaceous macromolecules, elicited in response to the presence of a foreign antigen, in the organism. Antibodies have the property of being capable of binding with antigen molecules in highly specific combinations. The binding is characterized by its high degree of specificity and low dissociation constant.
Normally, an animal has only those antibodies which are specifically directed against antigens to which it has been exposed in its environment. However, an animal can be induced to form antibodies against other antigens by artifically introducing them, for example by injection. Medical use is made of this phenomenon to immunize people against disease. It is also possible to cause a laboratory animal such as a rabbit or goat to make antibodies against specific substances. Such antibodies may be obtained from the blood of the animal and are exploited in highly specific assay techniques for the detection of the original antigenic substance. Virtually any protein can in principle be detected by means of an immunochemical reaction.
Immunochemical reactions have been exploited in a variety of ways: for the diagnosis of disease: for the identification of a specific infecting organism, as highly specific enzyme inhibitors, to determine the location of specific proteins in tissues and within cells, and for the quantitative measurement of specific proteins for which no chemical test is available. Since the reaction is a binding of one component (antigen) to another (antibody), there is no net change in the number of reactive groups as in an ordinary chemical reaction. Analysis of an immunochemical reaction therefore requires techniques for differentiating between bound and unbound components.
A variety of methods has been employed in the prior art for the measurement of immunochemical reactions. These include hemagglutination, latex particle agglutination, agar gel diffusion, complement fixation, counterelectrophoresis, and the use of antibodies tagged with fluorescent dyes or radioisotopes.
Hemagglutination and fluorescent antibody techniques have been applied in the detection of antibodies against Toxoplasma gondii. Toxoplasma is a protozoan parasite of man which lives primarily inside the cells of the host, so that the organism is difficult to detect by microscopic means. The vast majority of Toxoplasma infections are asymptomatic. However, an asymptomatically infected mother can pass the organism to the fetus, where Toxoplasma infection can cause a variety of birth defects: malformations, hydrocephalus, mental retardation, eye diseases often leading to blindness, and infant mortality. A simple, inexpensive screening test for pregnant mothers would be highly disirable as a step toward the eradication of congenital Toxoplasmosis. Although hemagglutination and fluorescent antibody techniques have been used in the diagnosis of Toxoplasma infection, the standard method has been the dye test. Serum antibody against Toxoplasma is detected in the dye test by taking advantage of the fact that live Toxoplasma cells are partially lysed in the presence of antibody. Lysed cells are distinguishable from unlysed cells by the addition of methylene blue which only stains intact organisms. In practice the test is complicated by the additional requirement for an accessory factor, thought to be complement, which must be obtained from the serum of Toxoplasma-free donors. Great attention to detail is required in carrying out the tests successfully. More significantly, the test is dangerous to laboratory workers since it involves the use of live Toxoplasma. A number of laboratory infections have resulted in individuals who have performed the test (see Jacobs, L., "Serodiagnosis of Toxoplasmosis", in Immunology of Parasitic Infections, S. Cohen and E. Sadun, eds., Blackwell Scientific Publications, 1976). An improved immunochemical method for detecting Toxoplasma infection is therefore highly desireable.
Although immunochemical methods generally used in the prior art can be highly sensitive, especially radioimmunoassay using radioisotopetagged antibody, the widespread usefulness of immunochemical assays has been limited by three factors:
(1) the need for a convenient and inexpensive method of separating bound immunochemically reacted components from unbound components,
(2) the need for a convenient and inexpensive way to measure the amount of immunochemical reactant bound, and
(3) the need for a procedure that can be carried out rapidly.
The first two of these difficulties have been overcome by recent advances in the prior art, the third is overcome by the present invention.
The first of these recent advances in the prior art is the development of techniques for coupling an immunochemically reactive antigen or antibody to an insoluble carrier material. An antibody immobilized on an insoluble carrier can, when exposed to a solution containing antigen, bind the antigen and render it, in turn, immobilized. The entire immunochemical reaction occurs on the carrier and the components which are bound on the carrier can be separated from the unreacted components by conventional techniques for separating solid phase materials from a liquid phase. For example, if the carrier is in the form of beads or finely divided powder, separation can be accomplished by centrifugation or decantation. Alternatively, the immobilized phase may be the inner surface of the reaction vessel itself, or it may be in the form of a sponge or porous matrix, so that separation may be carried out be simple decantation or by removal of the carrier, respectively.
The second advance has been the introduction of an enzyme-tagged antibody, which is a covalent conjugate of an antibody and an enzyme. Each retains its characteristic reactive properties: the antibody remains immunologically reactive and the enzyme retains its catalytic activity. When such a conjugate binds to an immobilized antigen, its presence can be detected through the activity of the coupled enzyme, after separating unbound conjugate by appropriate means.
These two techniques have been used in combination to develop a very sensitive type of assay termed an enzyme-linked immunosorbent assay. The invention which is the subject of this report embodies these basic techniques. For convenience, two types of enzyme-linked immunosorbent assay will be discussed and referred to as EL-1 and EL-2; see the diagram below: EQU EL-1: C--Ab--Ag--Ab-Enz EQU EL-2: C--Ag--Ab--AntiAb-Enz
C stands for an inert, insoluble carrier; PA1 Ab symbolizes antibody; PA1 Ag symbolizes antigen; PA1 C--Ab symbolizes carrier-bound antibody; PA1 C--Ag symbolizes carrier-bound antigen; PA1 Ab-Enz symbolizes the conjugate of an antibody with an enzyme; PA1 AntiAb-Enz symbolizes an antibody against immunoglobulin, conjugated with an enzyme.
EL-1 is a technique for detecting the presence of an antigen. Antibody against the antigen is immobilized on an insoluble carrier. The immobilized antibody is then exposed to an antigen-containing fluid. The immobilized antibody harvests the antigen from the solution by binding it in place. The carrier is then separated from the solution, washed free of contaminants, and exposed to a solution of the antibody conjugated to an enzyme. The principle of operation is that the conjugate is able to bind only at sites occupied by the antigen, and that the number of sites so occupied determines the amount of conjugate which can bind. Each site where antigen is bound is thus tagged with bound enzyme whose presence is manifested by its ability to catalyze a reaction. The rate of such reaction is proportional to the amount of enzyme present and becomes a direct measure of the amount of antigen bound.
In EL-2, the component to be detected is an antibody. Antigen is immobilized on the carrier. Binding of the antibody is then measured by the subsequent binding of an anti-immunoglobulin-enzyme conjugate. (See Wisdom, G. B., in Clinical Chemistry, Vol. 22, p. 1243 (1976).)
Antigen or antibody molecules may be immobilized on a solid carrier by a variety of methods known in the art, including covalent coupling, direct adsorption, physical entrapment and attachment to a protein-coated surface. For references describing this methodology, see Silman, I. H. and Katchalski, E. in Annual Review of Biochemistry, Vol. 35, p. 873 (1966); Melrose, G. J. H., in Review of Pure and Applied Chemistry, Vol. 21, p. 83, (1971); and Cuatrecasas, P. and Anfinsen, C. B., in Methods in Enzymology, Vol. 22, (1971).
The method of attachment to a protein-coated surface is disclosed by Lai et al. (German OS No. 2,539,657, U.S. Pat. No. 4,066,512). In this method, the internal and external surfaces of a microporous membrane are first coated with a water-insoluble protein such as zein, collagen, fibrinogen, keratin, glutelin, polyisoleucine, polytryptophan, polyphenylalanine, polytyrosine, or copolymers of leucine with p-amino phenylalanine. Such a coating renders the membrane capable of immobilizing a wide variety of biologically active proteins including enzymes, antigens, and antibodies. A microporous structure is defined as one having more than 50% of its total volume in the form of pores ranging in size from 25 nanometers to 25 micrometers, preferably from 25 nanometers to 14 micrometers. A pore size range from 25 nanometers to 5 micrometers is employed in most applications herein. Uncoated microporous membranes have as much as 70 to 75% of their volume as pore space. The pores permit liquid flow through the membrane. After being coated by zein, for example, the pore space is reduced 5 to 10% with the result that the structure retains its essential properties of having a high proportion of its volume as pore space and permitting liquid flow through the pores. The structure has a large surface area in contact with any solution contained within the pores.
Such a coated membrane, having immobilized antigen or antibody, provides a compact, easy to manipulate carrier for the immobilized antigen or antibody. Its integral structure permits removal of bound from unbound components by simple mechanical means.
A difficulty attending the use of microporous membranes as carriers for immobilized antigen or antibody is that these structures may adsorb proteins nonspecifically. Uncoated membranes of cellulose acetate and nitrate mixed esters can bind certain proteins and are also capable of exchanging bound for soluble protein. The physico-chemical basis for the binding is unclear. Certain proteins appear to bind more readily than others. As a result, assays based on binding a specific antigen, antibody or conjugate in the presence of a mixture of proteins can result in high background interference which may occur in an unpredictable manner.
A related difficulty is presented when coated membranes are used. Coated membranes, as disclosed by Lai, et al, are capable of binding proteins generally, but the binding is neither as selective nor as variable as that displayed by uncoated membranes. Consequently, the use of such membranes to immobilize antibody or antigen in an EL-1 or EL-2 assay may also result in high background interference due to the binding of unwanted protein species. These difficulties have been effectively surmounted by the present invention.